The invention relates to the preparation of a thromboplastin (TP) of specific factor VII sensitivity from mammalian tissue.
The term TP is to be understood, in accordance with DIN No. 58910, to mean an extract from mammalian tissue, which, in the presence of calcium ions, greatly shortens the coagulation time of blood plasma.
Thromboplastins are used diagnostically for the identification of coagulation abnormalities. The test used is referred to as Quick's test or the prothrombin time test. The events occurring in the case of a blood vessel injury, which ultimately lead to blood coagulation and to the closing of the vessel wound, are simulated by adding blood or blood plasma from the patient to tissue TP in the presence of calcium, and measuring the time it takes for a coagulum to form. The TP activates factor VII, which in turn brings about, through factors X and V, the formation of thrombin from prothrombin (factor II). Thrombin cleaves the fibrinogen to insoluble fibrin which participates in the closing of the wound. The time it takes after the addition of thromboplastin plus calcium for the formation of a clot is a measure of the concentration or activity of the coagulation factors involved.
Quick's test thus provides a summary knowledge of the concentration ratios of the coagulation enzymes (factors) involved. However, for the same Quick test values, the concentrations of the individual factors II, V, VII and X can be present in a great variety of ratios. Such alterations the concentration ratios of individual coagulation factors can be used, for example, by pathological changes (cirrhosis of the liver, intravascular clotting, vitamin deficiencies), or by medication, such as coumarin derivatives administered as oral anticoagulants. Differences become evident when the same test material is tested with thromboplastins from different manufacturers. These generally differ greatly in their sensitivity to individual coagulation factors, so that the same sample from a patient can have different prothrombin times when tested with TP from different manufacturers.
Therefore, it is desirable to achieve comparability between different thromboplastins. One way to accomplish this is standardization in reference to a particular factor sensitivity. There are numerous methods for the preparation of factor-sensitive TP. While the production of preparations sensitive to factors II, V and X presents no particular problem, difficulty still is encountered in the preparation of thromboplastin sensitive to factor VII.
It is assumed that a low factor-sensitivity of a thromboplastin is due to the fact that the preparation still contains traces of coagulation factors which are brought in through the blood content of the tissue used for the extraction of the thromboplastin. This assumption is supported especially by a finding reported in Thrombosis Hemostasis (Stuttgart) 1939, 592-599 (1978), according to which a thromboplastin that is especially sensitive to factor VII can be prepared from the brains of dogs with congenital factor VII deficiency.
Methods have already been described for the preparation of thromboplastins of special sensitivity to factor VII. For example, the desired effect is said to be achieved in accordance with DE-AS No. 2,556,493 by extracting the tissue directly with an alkali lye. It is highly probable that the thromboplastin is damaged by this method. This is also indicated by the fact that a relatively large number of adjuvants are used in this method for the stabilization of the thromboplastin product.
Another method is given in Biochem. Soc. Trans. 8, 133, (1980), namely the adsorption of factor VII onto barium sulfate. But according to the inventor's experience the thromboplastin largely precipitates together with the barium sulfate.
The isolation of thromboplastin from the tissue of animals with congenital factor VII deficiency is out of the question for industrial application merely for reasons of cost and the scarcity of the material.